Profiling the heterogeneous landscape of cell types and biomolecules is rapidly being adopted to address current imperative research questions. Precision medicine seeks advancements in molecular spatial profiling techniques with highly multiplexed imaging capabilities and subcellular resolution, which remains an extremely complex task. Surface-enhanced Raman spectroscopy (SERS) imaging offers promise through the utilization of nanoparticle-based contrast agents that exhibit narrow spectral features and molecular specificity. The current renaissance of gold nanoparticle technology makes Raman scattering intensities competitive with traditional fluorescence methods while offering the added benefit of unsurpassed multiplexing capabilities. Here, we present an expanded library of individually distinct SERS nanoparticles to arm researchers and clinicians. Our nanoparticles consist of a ∼60 nm gold core, a Raman reporter molecule, and a final inert silica coating. Using density functional theory, we have selected Raman reporters that meet the key criterion of high spectral uniqueness to facilitate unmixing of up to 26 components in a single imaging pixel in vitro and in vivo. We also demonstrated the utility of our SERS nanoparticles for targeting cultured cells and profiling cancerous human tissue sections for highly multiplexed optical imaging. This study showcases the far-reaching capabilities of SERS-based Raman imaging in molecular profiling to improve personalized medicine and overcome the major challenges of functional and structural diversity in proteomic imaging.

Multiplexing capabilities and sensitivity of surface-enhanced Raman spectroscopy (SERS) nanoparticles (NPs) are strongly dependent on the selected Raman reporter. These Raman-active molecules are responsible for giving each batch of SERS NPs its unique spectral fingerprint. Herein, we studied four types of SERS NPs, namely, AuNPs labeled with trans-1,2-bis(4-pyridyl)ethylene (BPE), 4,4′-bis(mercaptomethyl)biphenyl (BMMBP), 5-(4pyridyl)-1,3,4-oxadiazole-2-thiol (PODT), and 5-(4-pyridyl)-1H-1,2,4-triazole-3-thiol (PTT), and demonstrated that the best level of theory could be chosen based on inner products of DFT-calculated and experimental Raman spectra. We also calculated the theoretical spectra of these Raman reporters bound to Au 20 clusters to interrogate how SERS enhancement would affect their spectral fingerprint. Importantly, we found a correlation between B3LYP-D3 calculated and experimental enhancement factors, which opens up an avenue toward predicting which Raman reporters could offer improved sensitivity. We observed 0.5 and 3 fM limits of detection for BMMBP-and PTT-labeled 60 nm AuNPs, respectively.

Coloring dyes reveal potential as optical imaging contrast agents for cancer detection. Tattoo and Design created by artist Adam Sky.

Modern genomic sequencing efforts are identifying potential diagnostic and therapeutic targets more rapidly than existing methods can generate the peptide- and protein-based ligands required to study them. To address this problem, we have developed a microfluidic enrichment device (MFED) enabling kinetic off-rate selection without the use of exogenous competitor. We tuned the conditions of the device (bed volume, flow rate, immobilized target) such that modest, readily achievable changes in flow rates favor formation or dissociation of target–ligand complexes based on affinity. Simple kinetic equations can be used to describe the behavior of ligand binding in the MFED and the kinetic rate constants observed agree with independent measurements. We demonstrate the utility of the MFED by showing a 4-fold improvement in enrichment compared to standard selection. The MFED described here provides a route to simultaneously bias pools toward high-affinity ligands while reducing the demand for target-protein to less than a nanomole per selection.

Assays for chemical biomarkers are a vital component in the ecosystem of noninvasive disease state assessment, many of which rely on quantification by colorimetric reactions or spectrophotometry. While modern advances in microfluidic technology have enabled such classes of devices to be employed in medical applications, the challenge has persisted in adapting the necessary tooling and equipment to integrate spectrophotometry into a microfluidic workflow. Spectrophotometric measurements are common in biomarker assays because of straightforward acquisition, ease of developing the assay's mechanism of action, and ease of tuning sensitivity. In this work, 3D-printed, discrete microfluidic elements are leveraged to develop a model system for assaying hyaluronidase, a urinary biomarker of bladder cancer, via absorbance spectrometry of gold nanoparticle aggregation. Compared to laboratory microtiter plate-based techniques, the system demonstrates equivalent performance while remaining competitive in terms of resource and operation requirements and cost.

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